Ultramicroscopy 42-44 (1992) 1107-1112 North-Holland
Visualization of the algal polysaccharide carrageenan by scanning tunnelling microscopy I. Lee, E.D.T. Atkins 1 and M.J. Miles H.H. Wills Physics Laboratory, University of Bristol, Tyndall Avenue, Bristol BS8 1TL, UK Received 12 August 1991
Scanning tunnelling microscopy has been used to obtain images in the constant-current mode in air and moist conditions at molecular resolution for the kappa- and iota-carrageenan algal polysaceharides. The molecules were deposited from an aqueous solution onto a graphite substrate. The samples formed aligned nematic-like arrays and were also found as individual molecules. The molecular dimensions of width, height and repeat distance along the molecule were found to be close to the values previously determined by X-ray diffraction. The results support a molecular model based on a double-helix structure for carrageenan.
I. Introduction Structural investigations of polysaccharides using X-ray diffraction [1,2] show that these molecules can take up a variety of helical structures in the semi-crystalline state and as individual molecules. A number of naturally occurring polysaccharides are designed to form gels or liquid crystals . The family of carrageenan polysaccharides occur in red algae and are used extensively in the food industry as viscosity enhancers and gelling agents. The molecular structures of the macromolecule were deduced by analyses of X-ray diffraction patterns of fibres drawn from gels [3,4], The chemical structures for kappa- and iota-carrageenan are shown in fig. 1. They both consist of an idealized repeating disaccharide, linked 1,4 diequatorially and 1,3 diequatorially in an alternating fashion. Both molecules are polyelectrolytes with ionizable sulphate appendages; iota-carrageenan being the more heavily sulphated (see fig. la). Analyses of the X-ray fibre diffraction photographs [3-6] argue for a robust double-helix structure for both kappa- and 1 To whom correspondence should be addressed.
iota-carrageenan. The two chains, with the same chemical polarity, intertwine around a common axis with right-handed chirality and three-fold symmetry, each chain relatively half-staggered (in terms of translation). The pitch of the helix is 2.5 nm and the diameter ~ 1.3 nm as shown in fig. lb. The effect of relative rotations and translations away from exact half staggering have been discussed [5,6]. An aqueous solution (e.g. 2% by weight) of carrageenan at elevated temperature ( > 60 °C) is a viscous liquid. On cooling to room temperature it gels ( ~ 40 °C) and this process is reversible on reheating with some hysteresis. A molecular model for this reversible gelation mechanism was proposed by Rees , who envisaged individual chains existing above the gel melting point. On cooling (which can be induced by quenching if necessary) into the gel data the chains intertwine, for limited lengths, to form the particular doublehelix model described above. These double-helical regions are supposed to be the junction zones in the gel. A particular chain would intertwine with a number of different partners as it traces its way through the gel. The untwining, or more to the point, the retwining of chain segments, as a
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I. Lee et a L / Visualization of the algal polysaccharide carrageenan
1 J . CH°H.O
Fig. 1. (a) Idealized repeating disaccharide structures for (upper) kappa-carrageenan and (lower) iota-carrageenan. (b) Space-filling double-helix model of kappa-carrageenan based on X-ray diffraction results.
function of temperature, to form junction zones in the gel  is difficult to visualize from a topological standpoint. Although this original model for gelation has been modified to include aggregation of double-helical segments . within the junction zones, the single-chain to double-helix transition is still a necessary ingredient of the gelation mechanism and is used to account for the sudden increase in optical rotation on gelation. An alternative model based on side-by-side association of two carrageenan chains has been proposed  to overcome the serious topological problem of the Rees model [7,8], although this side-by-side model is not supported by X-ray diffraction studies on oriented films [4,6]. Thus it is important to have independent evidence for either a single chain or a double helix for carrageenan. The visualization of the carrageenan molecule using STM offers the opportunity to delineate between these two controversial structures for the molecule.
2. Experiment The STM used in these studies was manufactured by W.A. Technology (Cambridge, UK). It was mounted on an antivibration table and operated in air. Electrochemically etched tungsten
probes and mechanically cut gold probes were used. Images were recorded in the constant-current mode with typical tunnelling conditions in the range 0.1 to 1.0 nA and 600 to 1536 mV. Image acquisition times were between 44 and 100 S.
Samples of kappa- and iota-carrageenan, with molecular weights 3.9 × 105 and 4.3 × 105 respectively, were purchased from Sigma Chemical Company Ltd. Aqueous solutions were prepared at a concentration of 0.01%. The basic method used was to take a 1 ~1 drop of solution, place it on a highly oriented pyrolytic graphite substrate (ZYB grade from Union Carbide, USA) and allow to dry slowly. The deposited material on the graphite decreased in thickness towards the centre of where the drop of solution had been, and so by imaging at points along a radius from the centre, it was possible to study a range of sample thicknesses. Variations on this theme were to spray a fine mist of water on the sample after drying, or slightly before complete drying and before scanning with the tip. Scanning the tip in the wet edge area gave the highest-resolution images. One way for reducing molecular aggregation was to dip the clean graphite substrate into a 0.05% aqueous solution of polysaccharides at 20°C for 5 min to allow hydrophobic deposition to take place and then to
L Lee et al. / ~tsualization of the algal polysaccharide carrageenan
r e m o v e s u b s t r a t e , i n c l i n e d at a b o u t 10 °, a n d allow to dry. T h e s a m p l e s w e r e u n c o a t e d . W e a t t e m p t e d to d e p o s i t the s a m p l e s electrically trying to c a p i t a l i z e on the p o l y e l e c t r o l y t i c c h a r a c t e r o f the m o l e c u l e s b u t w e r e unsuccessful. Also addition of ammonium acetate or sodium
i o d i d e w e r e tried, since t h e y a r e k n o w n to supp r e s s m o l e c u l a r a g g r e g a t i o n , b u t w i t h o u t success. B e f o r e i m a g e s could be a t t r i b u t e d to the carr a g e e n a n s a m p l e s with sensible c o n f i d e n c e , t h e y w e r e c o m p a r e d with a c a t a l o g u e o f a r t i f a c t u a l
Fig. 2. STM images of kappa-carrageenan molecules on a graphite substrate: (a) size: 24.1 × 24.1 × 1.1 nm; (b) size: 9.2 x 9.2 x 1.3 nm). (c) Three-dimensional representation of the STM image (9.2 x 9.2 x 1.3 nm). Bias: 1539 mV; current: 0.1 nA.
1. Lee et al. / Visualization of the algal polysaccharide carrageenan
images and their associated current-voltage plots obtained from clean graphite surfaces. Many of those artifacts resemble long periodic molecules, and so considerable caution was exercised in scrutinizing the images we observed. We were particularly keen to obtain pictures of molecules lying at angles other than 90 ° or 0 ° to the scanning direction.
3. Results and discussion
The distribution of molecules deposited on the graphite substrate was found to be non-uniform. It was found necessary to locate regions sufficiently thin for successful STM imaging.
3.1. Kappa-carrageenan The highest-resolution images were obtained for kappa-carrageenan. Fig. 2a shows molecules which are a little wider, ~ 2 nm (compare with 1.3 nm), than expected but also a little lower, ~ 1 nm, and consistent with rod-like molecules being pulled down onto the substrate surface. By far the m o s t convincing feature is the periodicity of 1.4 nm (obtained by averaging a number of repeats) which relates to the expected value of 1.25 nm (half the pitch) found from X-ray diffraction. The dimensions are commensurate with expectations and the general features for a top surface topographic image of two intertwining chains match that expected for kappa-carrageenan. The
Fig. 3. STM image of iota-carrageenan molecules on a graphite substrate, (a) showing a substantial domain of aligned molecules, size: 50.5 x 50.5 x 0 . 3 nm; (b) three-dimentional representation of STM image, size: 17.5 x 17.5 x 1.6 nm; bias: 1536 mV; current: 1 nA; (c) line profile along the molecules of (b).
I. Lee et al. / Visualization o f the algal polysaccharide carrageenan
enlarged image (fig. 2) is consistent with a righthanded chirality (banding running at about 45 ° a direction clockwise to the molecular axis) but we judge that the resolution at this stage is not sufficient to constitute a proof. It should be compared with the projection of the space-filling model of kappa-carrageenan (fig. lb). The effect i v e lateral resolution of the STM is approximately [2(R + d)] 1/2, with tip radius R, and sample-to-tip distance d, so the tip radius has an important role in terms of resolving power. In these images we are not able to resolve the individual saccharide units or indeed confirm the three-fold nature of the individual helices. Fig. 2c is a three-dimensiohal image of kappa-carrageenan. Although the resolution is not sufficient to pick out the individual saccharide units, it is sufficient to follow the trace of the backbone helix. It is clear that the surface (edge in figures) periodicity is ~ 1.2 to 1.4 nm and not 2.4 to 2.6 nm which would be the expected periodicity of a single chain. Also the resolution is sufficient to rule out side-by-side single chains. Thus the images favour a double-helix molecule rather than any form of single-helix model that has been proposed. No images consistent with single helices were observed, even from solutions quenched from elevated temperature, indicating that all the images were of double-helical character.
3.2. Iota-carrageenan The resolution for iota is not quite so good as we observed for kappa but sufficient to see distinct inclined banding with an average periodicity of 1.54 nm (compare with the X-ray value of 1.3 nm) as shown in figs. 3a and 3b and the line profile (fig. 3c) of iota-carrageenan which was taken along the molecules from fig. 3b. The estimated width, 1.7 nm, and height, 1.4 nm, are consistent with the proposed molecular structure. Images were also obtained o f substantial domains of aligned molecules as shown in fig. 3a. Again the images favour a double helix with profile periodicity closer to the 1.3 nm value than to the value of 2.6 nm for a single helix.
4. Conclusions Images of both kappa- and iota-carrageenan have been obtained using STM. The photographs are consistent with the robust double helical molecular model based on X-ray diffraction results. The observed periodicity along the kappa-carrageenan molecule is measured at 1.4 nm, compared with the X-ray result of 1.3 nm and width and height indicate the molecule is pulled firmly onto the graphite substrate, squashing down and spreading the molecule slightly. The resolution is not quite as good for iota-carrageenan; the periodicity along the molecule is estimated to be 1.54 nm, compared with the X-ray result of 1.3 nm. The molecule seems slightly less distorted in terms of height and width compared with kappa-carrageenan. Although we have reported the visualization of STM images of polysaccharides before , the photographs of kappa-carrageenan here are of the highest resolution that has been reported to date for polysaccharides. It should be noted that although the results favour a doublehelix model for carrageenan, this should not be taken as support for the untwining/retwining model of Rees [7,8]. A more plausible model is that carrageenan is always a double helix and that the junction zones in the gel are aggregated of stiff double-helix segments  connected by more flexible double-helical molecules. This model has no topological problems and explains why single helices are never observed. The double-helix flexibility is a function of temperature and local associations.
Acknowledgements We wish to thank the Agriculture and Food Research Council for support for this work.
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1. Lee et al. / I,Tsualization o f the algal polysaccharide carrageenan
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